Mutational analysis of the β-subunit of yeast geranylgeranyl transferase I

The gene CAL1 (also known as CDC43 ) of Saccharomyces cerevisiae encodes the β subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant-enzymes encoded by cal1 , and cdc43-2 to cdc43-7 , expressed in bacteria, have hown that all of the mutant enzymes possess reduced activity, and that none shows temerature-sensitive enzymatic activities. Nonetheless, all of the cal1/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperative sensitive. The cal-1 mutation, located most proximal to the C-terminus of the protein, differs from the other cdc43 mutations in several respects. An increase in soluble Rholp was observed in the cal-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype of cal-1 is most efficiently suppressed by overproduction of Rholp. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious in cal-1 cells, but not in other cdc43 mutants or the wild-type strains. The cdc43-5 mutant cells accumulate Cdc42p in soluble pools and cdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among the cal1/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42258/1/438-252-1-2-1_62520001.pd

NADP-GDH and NAD-GDH activities of deletion mutants.

<p>NADP-GDH and NAD-GDH activities of deletion mutants.</p

Invasive growth of <i>gdh1</i>Δ, <i>gdh2</i>Δ, and <i>glt1</i>Δ mutants and effect of increasing concentrations of ammonium.

<p>Invasive growth was assayed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186028#pone.0186028.g001" target="_blank">Fig 1</a>. (A) HMP126 (WT), HMP135 (<i>gdh1</i>Δ), HMP128 (<i>gdh2</i>Δ), and HMP125 (<i>glt1</i>Δ) were grown on LNB medium. (B) HMP94 (WT), HMP137 (<i>gdh1</i>Δ), HMP133 (<i>gdh2</i>Δ), and HMP131 (<i>glt1</i>Δ) were grown on LNB medium and media in which 0.76 mM NH₄⁺ in LNB was replaced by 5 or 10 mM NH₄⁺.</p

<i>S</i>. <i>pombe</i> strains used in this study.

<p><i>S</i>. <i>pombe</i> strains used in this study.</p

Invasive growth defect of an <i>amt1</i>Δ mutant and effect of increasing concentrations of ammonium.

<p>Five-microliter aliquots of cell suspension (5 x 10⁴ cells) were spotted onto LNB medium and media in which 0.76 mM NH₄⁺ in LNB was replaced by 5 or 10 mM NH₄⁺. The plates were incubated at 30°C for 14 days and photographed before (upper panels) and after (lower panels) the cells were washed off the surface of the agar. Strains are HMP94 (WT) and HMP95 (<i>amt1</i>Δ). All images are shown at the same magnification, so that size of the cell patches reflects growth rate. It is evident that increasing concentrations of ammonium enhance not only invasive growth but also surface growth.</p

Filamentous invasive growth of mutants of the genes encoding ammonia-metabolizing enzymes in the fission yeast <i>Schizosaccharomyces pombe</i>

<div><p>The fission yeast <i>Schizosaccharomyces pombe</i> undergoes a switch from yeast to filamentous invasive growth in response to certain environmental stimuli. Among them is ammonium limitation. Amt1, one of the three ammonium transporters in this yeast, is required for the ammonium limitation-induced morphological transition; however, the underlying molecular mechanism remains to be understood. Cells lacking Amt1 became capable of invasive growth upon increasing concentrations of ammonium in the medium, suggesting that the ammonium taken up into the cell or a metabolic intermediate in ammonium assimilation might serve as a signal for the ammonium limitation-induced morphological transition. To investigate the possible role of ammonium-metabolizing enzymes in the signaling process, deletion mutants were constructed for the <i>gdh1</i>, <i>gdh2</i>, <i>gln1</i>, and <i>glt1</i> genes, which were demonstrated by enzyme assays to encode NADP-specific glutamate dehydrogenase, NAD-specific glutamate dehydrogenase, glutamine synthetase, and glutamate synthase, respectively. Growth tests on various nitrogen sources revealed that a <i>gln1</i>Δ mutant was a glutamine auxotroph and that a <i>gdh1</i>Δ mutant had a defect in growth on ammonium, particularly at high concentrations. The latter observation indicates that the NADP-specific glutamate dehydrogenase of <i>S</i>. <i>pombe</i> plays a major role in ammonium assimilation under high ammonium concentrations. Invasive growth assays showed that <i>gdh1</i>Δ and <i>glt1</i>Δ mutants underwent invasive growth to a lesser extent than did wild-type strains. Increasing the ammonium concentration in the medium suppressed the invasive growth defect of the <i>glt1</i>Δ mutant, but not the <i>gdh1</i>Δ mutant. These results suggest that the nitrogen status of the cell is important in the induction of filamentous invasive growth in <i>S</i>. <i>pombe</i>.</p></div

Growth of <i>gdh1</i>Δ, <i>gdh2</i>Δ, <i>gln1</i>Δ, and <i>glt1</i>Δ mutants on various nitrogen sources.

<p>Five-microliter aliquots of cell suspension (5 x 10⁴ cells) were spotted onto glucose minimal medium containing 10 mM ammonium (NH₄⁺), 10 mM glutamate (Glu), or 10 mM glutamine (Gln) (A), or 0.1, 1, 10, or 100 mM ammonium (B) as the nitrogen source. The plates were incubated at 30°C for 2 days before photographs were taken. Strains are the same set of strains as used for enzyme assays: HMP126 (WT), HMP135 (<i>gdh1</i>Δ), HMP128 (<i>gdh2</i>Δ), HMP141 (<i>gln1</i>Δ), and HMP125 (<i>glt1</i>Δ).</p